Early and late gut microbiota signatures of stroke in high salt-fed stroke-prone spontaneously hypertensive rats

The high salt-fed stroke-prone spontaneously hypertensive rat (SHRSP) is a suitable tool to study the mechanisms underlying stroke pathogenesis. Salt intake modifies the gut microbiota (GM) in rats and humans and alterations of the GM have previously been associated with increased stroke occurrence. We aimed to characterize the GM profile in SHRSPs fed a high-salt stroke-permissive diet (Japanese diet, JD), compared to the closely related stroke-resistant control (SHRSR), to identify possible changes associated with stroke occurrence. SHRSPs and SHRSRs were fed a regular diet or JD for 4 weeks (short-term, ST) or a maximum of 10 weeks (long-term, LT). Stroke occurred in SHRSPs on JD-LT, preceded by proteinuria and diarrhoea. The GM of JD-fed SHRSPs underwent early and late compositional changes compared to SHRSRs. An overrepresentation of Streptococcaceae and an underrepresentation of Lachnospiraceae were observed in SHRSPs JD-ST, while in SHRSPs JD-LT short-chain fatty acid producers, e.g. Lachnobacterium and Faecalibacterium, decreased and pathobionts such as Coriobacteriaceae and Desulfovibrio increased. Occludin gene expression behaved differently in SHRSPs and SHRSRs. Calprotectin levels were unchanged. In conclusion, the altered GM in JD-fed SHRSPs may be detrimental to gut homeostasis and contribute to stroke occurrence.

The SHRSP animal model represents a suitable experimental tool for studying the human disease.SHRSPs develop spontaneous hypertension and target organ damage, mainly renal and cerebrovascular damage, with aging 9 .Feeding SHRSPs with a high-salt/low-potassium diet (Japanese style diet, JD) accelerates the development of hypertensive target organ damage (vasculitis type) compared to the closely related control strain, the stroke-resistant SHR (SHRSR), despite similar BP levels 10 .Stroke occurrence is regularly anticipated in SHRSPs by the appearance of proteinuria, weight loss and often diarrhoea 11 .The latter may support the involvement of GM dysbiosis, inflammation and increased permeability in the higher stroke predisposition of this strain.Consistent with this, a recent work reported that the GM contributes to blood-brain barrier (BBB) disruption in SHRSPs 12 .In fact, BBB integrity was improved in SHRSPs fostered on WKY dams compared to SHRSPs fostered on their own dams, suggesting that environmental factors such as the GM of the foster dam may regulate it 12 .Of note, high salt intake, which favours stroke in SHRSP, caused changes in the GM and induced hypertension in normotensive rats 13 .High salt intake also induced significant changes in the human GM 14 .However, evidence on the possible pathogenic contribution of dysbiosis to the higher stroke predisposition of high salt fed SHRSPs is still lacking.In the present study, we aimed to explore this issue and to identify the alterations of the GM induced by the high-salt stroke permissive diet (JD) in SHRSPs compared to SHRSRs, both before stroke appearance and at the time of stroke occurrence.We also assessed inflammatory status by measuring the serum levels of calprotectin and the gut barrier integrity by assessing gene expression of the tight-junction proteins zonulin (ZO-1) and occludin (Ocln).

Phenotypic parameters of SHRSRs and SHRSPs after short-term (4 weeks) JD feeding
Body weight (BW) increased significantly in a comparable manner in SHRSRs and SHRSPs (p < 0.001, two-way ANOVA) over 4 weeks (short term, ST) of JD (JD-ST) or control diet (regular diet, RD) feeding (Fig. 1a,b), but it was generally higher in SHRSR animals compared to SHRSP animals upon the same diet.However, there was not a significant difference in BW between different rat strain fed with the same diet for the same number of weeks.Initial weights of animals are available in Supplementary Table S1.Proteinuria levels were significantly increased in SHRSPs JD-ST compared to RD-fed SHRSPs, RD-fed SHRSRs and SHRSRs JD-ST (p < 0.001, twoway ANOVA) (Fig. 1c).Systolic blood pressure (BP) was not significantly different among the four groups (Fig. 1d).No signs of stroke occurred during the 4-week period in either JD or RD-fed rats.Renal histology revealed the presence of damage in SHRSPs as previously reported [15][16][17] (Fig. 1e).

Phenotypic parameters of SHRSRs and SHRSPs on long-term JD feeding
The increase in BW was different between the two rat groups fed long-term (LT) JD, being significantly higher in SHRSRs JD-LT than in SHRSPs JD-LT from week 5 of JD (p < 0.001, two-way ANOVA followed by Tukey post-hoc comparison) (Fig. 2a).Proteinuria levels significantly increased in SHRSPs JD-LT after 6 weeks of JD (p < 0.001, one-way ANOVA followed by Tukey post-hoc comparison).In contrast, proteinuria levels did not change in SHRSRs JD-LT (Fig. 2b).Systolic BP increased significantly over time in both SHRSRs JD-LT and SHRSPs JD-LT (Fig. 2c).
Stroke started to occur in SHRSPs JD-LT after 6 weeks of JD feeding (25% incidence), and its incidence increased to 100% at week 9 of JD feeding (Fig. 3).Stroke was manifested by signs of limb paresis/hemiparesis, epileptic attacks, and sudden death, and it was always preceded by diarrhea and proteinuria.Stroke never occurred in SHRSRs JD-LT 10,11 .

Gut microbiota profile in JD-fed SHRSRs and SHRSPs
The large intestinal microbiota was profiled in SHRSRs and SHRSPs after 4 weeks of RD or JD feeding (JD-ST) and after long-term JD feeding (JD-LT).No differences in alpha diversity were found between the study groups (Supplementary Fig. S1).Similarly, no segregation between groups was found in the PCoA plots of unweighted and weighted UniFrac distances (Supplementary Fig. S1).
Notably, JD-fed SHRSRs and SHRSPs differed in the proportions of several taxa in both ST and LT (Fig. 5a and Supplementary Figs.S2 and S3).The SHRSP JD-ST group was characterized by a significantly higher relative abundance of the phyla Bacteroidetes and Proteobacteria, and a lower relative abundance of Firmicutes compared to SHRSRs JD-ST (p < 0.05, Mann-Whitney U test), suggesting an early strain-dependent high-level disruption of the GM.SHRSPs JD-ST also showed a significant overrepresentation of the family Streptococcaceae (and its genus Streptococcus) and an underrepresentation of Lachnospiraceae (especially Blautia) compared to the SHRSR counterpart (p < 0.05).It should be noted that Blautia proportions were significantly higher in both SHRSRs JD-ST and JD-LT compared to RD-fed SHRSRs (p < 0.05), suggesting a dietary and strain-related effect.The main discriminating taxa between the two rat strains JD-LT were the phylum Actinobacteria and the families Coriobacteriaceae, Erysipelotrichaceae (and its genus Allobaculum) and Desulfovibrionaceae (and Desulfovibrio), which were enriched in SHRSPs.In contrast, the families Peptostreptococcaceae, Alcaligenaceae (and Sutterella), Helicobacteraceae and Turicibacteraceae (and Turicibacter), and the genera [Prevotella], Candidatus Arthromitus, Lachnobacterium and Faecalibacterium were significantly enriched in SHRSRs (p < 0.05).Again, diet and straindependent patterns were found for some taxa, namely Actinobacteria, Erysipelotrichaceae and Sutterella, whose increases were also significant compared to the RD-fed counterparts (p < 0.05).See Fig. 5a and Supplementary Figs.S2 and S3 for all significant differences between the study groups, including those between the two RD-fed rat strains.When looking for correlations between relative taxon abundance and host metadata, we found that BP was negatively correlated with the families Porphyromonadaceae, S24-7 and [Paraprevotellaceae] (tau ≤ − 0.244, p ≤ 0.01, Kendall rank correlation test), while a positive correlation was found with Enterobacteriaceae and Spirochaetaceae (tau ≥ 0.215, p ≤ 0.04) (Fig. 5b).Positive associations were also observed for Bifidobacteriaceae and Coriobacteriaceae with proteinuria levels (tau ≥ 0.208, p ≤ 0.03), while Clostridiaceae and Christensenellaceae were negatively correlated (tau ≤ − 0.217, p ≤ 0.03) (Fig. 5c).

Serum calprotectin
Serum calprotectin levels ranged from 77.9 to 775.9 ng/ml (Fig. 6).No statistically significant difference was observed between the study groups.Three out of the 50 samples analyzed were excluded because they were below the detection limit.

ZO-1 and Ocln relative gene expression in the small intestinal mucosa
RT-qPCR of ZO-1 yielded measurable cycle threshold (Ct) levels in 88 out of 110 wells, while Ocln expression analysis yielded 104 out of 110 measurable Ct levels (samples in duplicate).
No changes in ZO-1 mRNA expression levels were detected among the two strains and the two dietary regimens (Fig. 7a-c, t-test).In contrast, Ocln mRNA levels were significantly lower in RD-fed SHRSPs compared to RD-fed SHRSRs (p < 0.001) (Fig. 7d) and significantly increased in SHRSPs JD-ST and JD-LT compared to RD-fed counterparts (p < 0.001) (Fig. 7f, two-way ANOVA).In contrast, Ocln levels were not higher in JD-fed SHRSRs compared to RD-fed SHRSRs (Fig. 7e).Two-way ANOVA confirmed an interaction between diet and strain both after 4 weeks of diet (RD vs. JD-ST) and at the end of the experiment (RD vs. JD-LT, p < 0.001).

Discussion
The present study provides the first GM profiling in SHRSPs fed with a high-salt stroke-permissive dietary regimen compared to the closely related stroke-resistant control (SHRSR).The JD-fed SHRSP represents a suitable experimental tool to investigate molecular mechanisms underlying stroke predisposition with a potential relevance for the human disease.Furthermore, based on the available literature, high salt intake elicits alterations in the GM of rats 13 , and this happens in a strain dependent manner for certain taxa.
In this study we found that JD-fed SHRSPs exhibited both early and late alterations in the GM composition compared to SHRSRs.Specifically, after short-term JD feeding, a critical time window that triggers the following stroke events, SHRSPs showed a high-taxonomic level rearrangement involving the major phyla Firmicutes and Bacteroidetes, with an overrepresentation of Streptococcaceae (and Streptococcus) and an underrepresentation of Lachnospiraceae (especially Blautia).Of note, Streptococcus has been previously found to be overabundant in hypertensive subjects and it has been proposed as a cause of increased vasoconstriction through increased serotonin production and consequent excessive sympathetic activation 18 .Some Streptococcus spp.have also been found to be increased in the GM following stroke 19 , strengthening their association with vascular events and outcomes.The variation observed inSHRSPs with regard to Lachnospiraceae members is also consistent with the available literature in hypertensive subjects 20 .Furthermore, Lachnospiraceae members are undoubtedly associated with healthy dietary patterns since their main metabolites, short-chain fatty acids, have been shown to regulate BP, to improve microcirculation, and to exert anti-inflammatory effects 21,22 .
Notably, after long-term JD feeding, the above-mentioned changes were no longer observed, suggesting a possible predominant effect of the diet.However, SHRSPs and SHRSRs still exhibited distinct microbiota compositional features, indicating the persistence of strain-specific patterns that may correlate with stroke  www.nature.com/scientificreports/occurrence upon prolonged JD feeding.These features included an increase of Actinobacteria (and of its family Coriobacteriaceae), Erysipelotrichaceae (and of its genus Allobaculum) and Desulfovibrionaceae (and Desulfovibrio) in SHRSPs.In contrast, Peptostreptococcaceae, Alcaligenaceae (and Sutterella), Helicobacteraceae, Turicibacteraceae (and Turicibacter), [Prevotella], Candidatus Arthromitus, Lachnobacterium and Faecalibacterium increased in SHRSRs.Again, few variations were expected, such as the SHRSP-related decrease in the shortchain fatty acid producers Turicibacter, Lachnobacterium and Faecalibacterium, and the SHRSP-related increase in the pathobionts Coriobacteriaceae and Desulfovibrio.In particular, Coriobacteriaceae members were found to be overrepresented in patients with symptomatic atherosclerosis 23 .Notably, the relative abundance of Coriobacteriaceae was positively correlated with proteinuria levels in our cohort 24,25 .Moreover, Desulfovibrio was found in hypertensive subjects and in patients with large-artery atherosclerotic ischemic stroke and transient ischemic attack 19,26 .Interestingly, the Desulfovibrio pathogenicity is likely related to excessive production of H 2 S, which could be deleterious in ischemic stroke by impairing mitochondrial function and inducing neurotoxicity 27 .The latter effects may be consistent with the known mitochondrial dysfunction observed in SHRSPs 28 and may support the development of the small vessel cerebrovascular disease of the strain.On the other hand, the increase of Allobaculum in SHRSPs contrasts with the available evidence, which shows an association between this mucindegrading taxon and lower BP in SHRs 29 , as well as improved hypertension-induced endothelial dysfunction 30 .Altogether, our study highlighted a different behavior of certain taxa in the SHRSP vs. SHRSR strains upon the stroke-permissive diet, and some of these variations could be consistent with a contributory pathogenetic role.Based on our current findings, future studies may assess the impact of either microbiota depletion through antibiotic treatment or fecal transplantation on stroke occurrence in SHRSPs.
Regarding intestinal permeability, the available evidence in stroke pathogenesis is limited and contradictory.Some studies in mice have shown that the small intestinal permeability is increased because of decreased expression of tight-junction proteins (ZO-1 and Ocln) 31 , while others have shown that stroke does not alter intestinal shape and function 32 .Low levels of ZO-1 and Ocln were found in SHRSRs with established hypertension compared with normotensive controls (Wistar Kyoto rats) 33 .In our study, ZO-1 expression did not change between the two rat strains on the two diets.In contrast, Ocln expression was significantly lower in SHRSPs compared to SHRSRs upon RD and increased in a time-dependent manner only in JD-fed SHRSPs, suggesting a strain-dependent interaction with the diet.The increase of Ocln could be explained as a compensatory mechanism to the diet-induced intestinal derangements, likely due to increased proteasomemediated degradation 34 , and it may support the involvement of reduced gut permeability in stroke pathogenesis.
Inflammation is recognized as an important element in the pathogenesis of ischemic stroke.In previous studies, circulating calprotectin levels were found to be elevated in acute ischemic stroke 35 and after ischemic stroke onset 36 , possibly predicting 3-month mortality 37 .Although SHRSPs carry a vasculitis-like cerebrovascular damage, no strain/diet-related changes in serum calprotectin levels were detected in our study.
The main limitations of the present study include: (i) the small number of animals used; (ii) the lack of more frequent longitudinal sampling to reconstruct the temporal dynamics of changes in the microbiota; (iii) the associative nature of the experimental design, which did not involve mechanistic investigation; and (iv) the use of 16S rRNA amplicon sequencing instead of shotgun metagenomics, which did not allow high-resolution taxonomic and functional profiling.
In conclusion, our study showed that the GM of JD-fed SHRSPs undergoes both early and late compositional changes that differ from their SHRSR counterparts, some of which may be detrimental to gut homeostasis and may contribute to the occurrence of stroke.These hypothesis-generating findings need to be confirmed in larger sample sets, possibly collected over time, and analysed using different approaches, including omics and functional analysis, to elucidate the role of the identified microbial signatures in stroke pathogenesis.A deeper understanding of the underlying molecular mechanisms may pave the way for future GM-targeted preventive and therapeutic strategies against hypertension and its consequences, including stroke.

Experimental design and sampling
Male SHRSPs (n = 27) and SHRSRs (n = 28) were housed in the animal facility at the Neuromed Institution (Pozzilli, Italy).Rats were kept 2 or 3 per cage under controlled conditions of light, temperature, and humidity.For the first six weeks of life, the rats received a standard rat chow diet and had free access to water.Then, rats of both strains were randomly divided into two subgroups: the first subgroup (SHRSR, n = 9 and SHRSP, n = 7) received a control diet (regular diet -RD, containing 22% protein, 2.7 mg/g Na + , 7.4 mg/g K + , 0.05 mg/g methionine), and the second subgroup (SHRSR, n = 19 and SHRSP, n = 20) received a high-salt/low-potassium Japanese style diet -JD, containing 17.5% protein, 3.7 mg/g Na + , 6.3 mg/g K + , 0.03 mg/g methionine, with the addition of 1% NaCl in the drinking water.Both RD and JD were provided by Lab.Piccioni, Milan, Italy.After 4 weeks of either RD or JD (short-term, ST), rats of both strains were sacrificed (SHRSP RD, n = 7; SHRSP JD-ST, n = 7; SHRSR RD, n = 9; SHRSR JD-ST, n = 8).The remaining rats continued JD (long-term, LT) until week 10 (SHRSR JD-LT, n = 11) or until death by stroke, which occurred between week 6 and week 9 (SHRSP JD-LT, n = 13) (Fig. 8).Rats were regularly monitored throughout the experiment for BW, systolic BP levels (measured by tail-cuff sphygmomanometry), proteinuria levels, diarrhoea occurrence, and signs of stroke (paresis/hemiparesis of limbs, epileptic attacks, sudden death).At the end of the appointed dietary regimen or at the time of stroke, the animals were sacrificed, urine and serum samples were collected, and the large and small intestines were removed, washed in PBS, and stored at -80°C until analysis.A portion of the large intestine was cut longitudinally, and the content was collected and stored at -80 °C for microbiota profiling.A portion of the small intestine was cut longitudinally, the content was removed, the lumen was washed with PBS and the mucosa was collected by scraping the intestinal walls before storage at -80 °C until mRNA analysis.Rats were sacrificed by decapitation under anesthesia (isoflurane, 5% for induction, and 2% for maintenance).Histological examination of kidneys of SHRSRs and SHRSPs was performed as previously reported by Rubattu et al. 38 .All animal studies were performed in accordance with approved protocols by the IRCCS Neuromed Animal Care Review Board and the "Istituto Superiore di Sanità" (ISS permit number: 1086/2020) and were conducted according to EU Directive 2010/63/EU for animal experiments.The study is reported in accordance with the ARRIVE guidelines (https:// arriv eguid elines.org).

Gut microbiota analysis: microbial DNA extraction and 16S rRNA amplicon sequencing
Microbial DNA was extracted from about 200 mg of large intestine content using the QIAamp DNA Stool Mini Kit (Qiagen, Hilden, Germany) with minor modifications as described in Costabile et al. 39 .Briefly, samples were suspended in 1.4 ml of ASL buffer and incubated at 95 °C for 5 min.Samples were then treated two times in a FastPrep-24 instrument (MP Biomedicals, Irvine, CA, USA) at 5.5 movements per sec for 45 s, after the addition of four 3-mm glass beads and 0.5 g of 0.1-mm zirconia beads (BioSpec Products, Bartlesville, OK, USA), and kept on ice for 5 min between treatments.After centrifugation at 15,000×g for 1 min at room temperature, half of the InhibitEX tablet was added to 1.2 ml of the supernatant and samples were mixed vigorously before incubation at room temperature for 2 min.DNA extraction was then continued following the manufacturer's instructions (Qiagen).DNA concentration and quality were assessed using a NanoDrop ND-1000 spectrophotometer (NanoDrop Technologies, Wilmington, DE, USA).
Libraries of the V3-V4 hypervariable regions of the 16S rRNA gene were prepared using the 341F and 785R primers with Illumina adapter overhang sequences 40 and following the "16S Metagenomic Sequencing Library Preparation" protocol (Illumina, San Diego, CA, USA).The final libraries, indexed and purified, were pooled at an equimolar concentration, denatured, and diluted to 5 pM before sequencing on an Illumina MiSeq platform with a 2 × 250 bp paired-end protocol according to the manufacturer's instructions (Illumina).

Serum calprotectin levels
To evaluate the inflammatory status of the gut, serum calprotectin levels were assessed using the Rat Calprotectin (CALP) ELISA Kit (Cusabio, Houston, TX, USA) according to the manufacturer's protocols.The reported kit sensitivity is 15.6 ng/ml.

RNA isolation and reverse transcription-quantitative PCR (RT-qPCR) for ZO-1 and Ocln
To investigate the integrity of the gut epithelial barrier, ZO-1 and Ocln gene expression levels were measured in the small intestine.Total RNA was extracted from 50 mg of small intestinal mucosa.The tissue was added with 1 ml of TRIzol™ Reagent (Life Technologies, Carlsbad, CA, USA) and three 3-mm glass beads (BioSpec Products), and homogenized twice in a tissue lyser (Tissue Lyser LT, Qiagen) at 50 Hz for 30 s.After the addition of 200 µl Figure 8. Diagram of experimental groups and samplings.SHRSR and SHRSP animals were sacrificed after 4 weeks of regular diet (RD) (SHRSR RD and SHRSP RD groups, respectively), or after 4 weeks (short term, ST) of Japanese Diet (JD) (SHRSR JD-ST and SHRSP JD-ST groups, respectively).JD-fed rats continued JD (long-term, LT) for 10 weeks before being sacrificed (SHRSR JD-LT group) or until death by stroke, which occurred between week 6 and week 9 (SHRSP JD-LT group).Serum, large and small intestines, and kidneys were collected after sacrifice.Body weight of each animal was measured weekly from the beginning of the diet until sacrifice, and blood pressure and proteinuria were measured during the 4th, 6th, 8th and 10th week of the diet.Stroke onset was continuously monitored throughout the experiment.The figure was created using icons from https:// www.freep ik.com/.

Figure 1 .
Figure 1.Phenotypic parameters of SHRSRs and SHRSPs after short-term (ST, 4 weeks) regular diet (RD) or Japanese diet (JD).Body weight change (%) during 4 weeks of RD (a) or JD (b) feeding in the two rat strains.(c) Proteinuria levels after 4 weeks of RD or JD feeding in the two rat strains.(d) Systolic blood pressure levels at the same experimental times.Two-way ANOVA followed by Tukey post-hoc comparison.In panels (a) and (b), only different rat strains at the same time point were statistically compared.SHRSR RD, n = 8 in panel a, n = 9 in panel (c) and (d); SHRSP RD, n = 7; SHRSR JD-ST, n = 7; SHRSP JD-ST, n = 7. (e) Azan Masson's trichrome staining showed a significant increase in perivascular (*) and glomerular ( <) fibrosis in SHRSPs, but not SHRSRs, fed JD for 4 weeks compared with the corresponding controls fed RD.Bar graphs show quantification of fibrosis as percentage of blue.Data are mean + /-standard error of the mean (SEM).Mann-Whitney twotailed test.*p < 0.05, **p < 0.01, ***p < 0.001.

Figure 3 .
Figure 3. Stroke occurrence in SHRSRs and SHRSPs upon long-term JD feeding.Percentage of stroke events in the two rat strains upon JD-LT treatment.The comparison between the two strains was significantly different (Log-rank and Mann-Whitney test, p < 0.0001).For the number of animals included in this analysis, see Fig.2.

Figure 4 .
Figure 4. Phylum and family-level gut microbiota composition of SHRSRs and SHRSPs after 4 weeks of regular diet (RD) or Japanese diet (JD-ST), and after long-term JD feeding (JD-LT).Bar plots showing the relative abundance of major phyla (a) and families (b) in the large intestine of the two rat strains after 4 weeks of RD or JD feeding (JD-ST) and after long-term JD feeding (JD-LT).Only taxa with a relative abundance > 0.1% in at least 3 samples are shown.SHRSR RD, n = 9; SHRSP RD, n = 7; SHRSR JD-ST, n = 8; SHRSP JD-ST, n = 7; SHRSR JD-LT, n = 11; SHRSP JD-LT, n = 13.

Figure 5 .
Figure 5. Family-level gut microbiota signatures of SHRSRs and SHRSPs after 4 weeks of regular diet (RD) or Japanese diet (JD-ST), and after long-term JD feeding (JD-LT), and correlations with blood pressure and proteinuria levels.(a) Heatmap showing Ward-linkage clustering based on Pearson's correlation coefficients of the relative abundance of gut microbiota families of the two rat strains after 4 weeks of RD or JD feeding (JD-ST) and after long-term JD feeding (JD-LT).Only taxa with a relative abundance > 0.1% in more than 2 samples are shown.See Supplementary Fig. S2 for further details.Scatter plots of correlation between relative abundance of families and (b) blood pressure (BPmmHG) and (c) proteinuria levels in SHRSRs and SHRSPs after RD or JD-ST/JD-LT.Only statistically significant correlations (p ≤ 0.05) based on Kendall rank correlation test with absolute tau ≥ 0.2 are shown.